Molecular beacons are single-stranded oligonucleotide hybridisation probes that form a stem-and-loop structure. The loop contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary sequences located on either side of the probe sequence. A fluorophore is covalently linked to the end of one arm and a quencher to the end of the other arm. In the absence of targets, the probe does not fluoresce, because the stem places the fluorophore so close to the non-fluorescent quencher that they transiently share electrons, eliminating the ability of the fluorophore to fluoresce. When the probe encounters a target molecule, it forms a probe-target hybrid that is longer and more stable than the stem hybrid. The rigidity and length of the probe-target hybrid precludes the simultaneous existence of the stem hybrid. Consequently, the molecular beacon undergoes a conformational reorganization that forces the stem hybrid to dissociate and the fluorophore and the quencher to move away from each other (FIG. 1). For a general overview on the scientific and patent literature of molecular beacons, see Tyagi, S.; Kramer, F. R., Nature Biotechnology 1996, 14, 303-308, and www.molecular-beacons.org, a website of Public Health Research Institute, Newark (N.J.), USA.
Molecular beacons can be used as amplicon detector probes in diagnostic assays. Because non-hybridised molecular beacons are non-fluorescent, it is not necessary to isolate the probe-target hybrids to determine the number of amplicons synthesized during an assay. Molecular beacons are added to the assay mixture before carrying out gene amplification and fluorescence is measured in real time. Furthermore, the use of molecular beacons provides an additional level of specificity. Because it is very unlikely that false amplicons or primer-dimers possess target sequences for the molecular beacons, the generation of fluorescence is exclusively due to the synthesis of the intended amplicons.
Molecular beacons with differently coloured fluorophores can be synthesized. This enables assays that simultaneously detect different targets in the same reaction. For example, multiplex assays contain a number of different primer sets, each set enabling the amplification of a unique gene sequence, e.g. from different pathogenic agents. A corresponding number of molecular beacons can be present, each containing a probe sequence specific for one of the amplicons, and each labelled with a fluorophore of a different colour. The colour of the resulting fluorescence identifies the pathogenic agent in the sample and the number of amplification cycles required to generate detectable fluorescence provides a quantitative measure of the number of target sequences present. Moreover, due to the inherent design of gene amplification assays, the use of molecular beacons enables the detection of a rare pathogen in the presence of a much more abundant pathogen.
The stem region of a molecular beacon is particularly critical to the successful application of a molecular beacon. The nucleobases of the stem can interact (or pair) with the nucleic acid target in an undesirable way. This property leads to hybridisation to wrong nucleotide sequences and thus reduces the specificity of the method.
In patent applications WO03/051901, WO03/052132, WO03052133 and WO03052134 (Unest A/S, Christensen, U. B., and Pedersen, E. B.) the use of polyaromatic or heteroaromatic building blocks in oligonucleotides are described. Hairpin oligonucleotides comprising such building blocks are described and claimed, but the authors did not describe the potential of using such building blocks in molecular beacons for stabilizing the stem region as is described in the present invention.
The principle of detecting a target polynucleotide using an oligonucleotide probe comprising substituents able to form an excimer under particular conditions is, for example, described in U.S. Pat. No. 5,332,659 (Kidwell, D. A.). U.S. Pat. No. 5,925,517 (Tyagi, S. et al.) and related patents U.S. Pat. No. 6,103,476; U.S. Pat. No. 6,150,097; and U.S. Pat. No. 6,037,130 describe hybridisation probes with label pairs that can be used to generate a signal when the labels are in close proximity, e.g. FRET pairs consisting of a fluorescent label and a quencher label.